This is a renewal application for a project previously funded for a total of 9 years. Signal transduction via guanine nucleotide binding proteins (G proteins) is crucial in regulation of cardiovascular , neural and endocrine function. More recently a role has been defined for heterotrimeric G proteins in cell growth and regulation as well. In spite of impressive structural advances, our understanding of the dynamics is very limited. To assist in the design of activators or inhibitors of G protein function and to better understand the steps in G protein activation the applicant proposes to study the kinetic mechanisms of G protein activation and deactivation by the use of several newly developed biophysical techniques. Specifically the applicant proposes to use fluorescence resonance energy transfer and flow cytometery to define the multiple steps between binding of the ligand to the receptor and dissociation of G protein subunits. The applicant proposes four specific aims: 1) identification of G protein conformational changes and subunit association/ dissociation equilibria by direct activators 2) rapid kinetic examination of the rates and mechanisms of G protein deactivation by RGS 3) examination of the individual steps in G protein activation by receptors and 4) optimization of fluorescence resonance energy transfer methods to study protein-protein interactions within the cell.